Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

Abstract

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched antisense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells. Int.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells

“…A possible explanation for this phenomenon could be the frequent mutations of K-ras observed in pancreatic (Bos, 1989) and colorectal carcinomas (Bos et al, 1987) that might impact on the ability of mda-7/IL-24 to induce apoptosis in these two cancer cell types (Su et al, 2001;Lebedeva et al, 2005b;Lebedeva et al, 2006). Several studies suggest that downregulation of K-ras expression may be of benefit in the treatment of pancreatic (Aoki et al, 1997;Kita et al, 1999) and colorectal (Mukhopadhyay et al, 1991;Shirasawa et al, 1993;Cochet et al, 1998) cancers. These findings imply that this oncogene may provide a viable target for inactivation, thereby improving response to other therapies.…”

DOC-2/hDab2 Expression Is Up-Regulated in Primary Pancreatic Cancer but Reduced in Metastasis

“…Next, we investigated whether suppressing K-ras expression would influence DOC-2 transcription in those two pancreatic cancer cell lines. To this end, a mutationmatched K-ras antisense oligonucleotide approach was performed as described previously (Kita et al, 1999). First, Western blot analysis was carried out to confirm that the K-ras antisense oligonucleotides effectively decrease K-ras p21 protein levels (Fig.…”

“…Transfection experiments were carried out as described previously in detail (Kita et al, 1999). Briefly, lipofectamine was gently mixed with the oligonucleotides in a serum-free cell culture medium and incubated at room temperature for 45 minutes to form complexes.…”

“…The final concentrations of lipofectamine and oligonucleotides were 10 g/ml and 1 M, respectively. The design of the specific K-ras sense and antisense oligonucleotides has been described previously at length (Kita et al, 1999).…”

Ras-dependent carbon metabolism and transformation in mouse fibroblasts

“…Various strategies for the downregulation of Ras pathway have been reported: treatment of antibodies to K-Ras (Cochet et al, 1999;Russell et al, 1999), incorporation of a dominantnegative K-Ras mutant into target cells (Takeuchi et al, 2000), expression of an antisense specific to K-ras (Monia et al, 1992;Kita et al, 1999), retroviral delivery of a ribozyme (Kijima et al, 2004), short interfering RNAs (Brummelkamp et al, 2002) and utilization of compounds able to block the Ras-Raf interaction (Kato-Stankiewicz et al, 2002). We developed a genetically defined experimental system that allowed us to show that expression of a GEF dominant-negative mutant (that we call GEF-DN) downregulates Ras activity both in vitro (Vanoni et al, 1999) and in vivo on the basis of morphology, anchorage-independent growth and reduction of Ras-dependent tumor formation in nude mice (Bossu' et al, 2000).…”